Safety, Immunogenicity, and Efficacy of a Plasmodium falciparum ...

Vol. 60, No. 5

INFEcrION AND IMMUNITY, May 1992, p. 1834-1839

0019-9567/92/051834-06$02.00/0

Copyright © 1992, American Society for Microbiology

Safety, Immunogenicity, and Efficacy of a Plasmodium falciparum Vaccine Comprising a Circumsporozoite Protein Repeat Region Peptide Conjugated to Pseudomonas aeruginosa Toxin A L. F. FRIES,"* D. M. GORDON,2 I. SCHNEIDER,2 J. C. BEIER,3 G. W. LONG,1 M. GROSS,4 J. U. QUE,5 S. J. CRYZ,S AND J. C. SADOFF2

Center for Immunization Research' and Department of Immunology and Infectious Disease,3 Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205; Walter Reed Army Institute of Research, Washington, D.C. 203072; SmithKline Beecham, Philadelphia Pennsylvania 191014; and Swiss Serum and Vaccine Institute, Berne, Switzerland' Received 21 November 1991/Accepted 24 February 1992

Twenty-one malaria-naive volunteers were immunized with a vaccine consisting of a 22-kDa recombinant peptide (R32LR), derived from the repeat region of Plasmodium falciparum circumsporozoite (CS) protein, covalently coupled to detoxified Pseudomonas aeruginosa toxin A. Nineteen volunteers received a second dose of vaccine at 8 weeks, and eighteen received a third dose at 8 to 12 months. The vaccine was well tolerated, with only one volunteer developing local discomfort and induration at the site of injection which limited function for 48 h. The geometric mean anti-CS immunoglobulin G antibody concentration 2 weeks after the second dose of vaccine was 10.6 ,ug/ml (standard deviation = 3.0 ,ug/ml). Eleven volunteers (52%) developed anti-CS antibody levels of >9.8 ,ug/ml, the level measured in the one volunteer protected against P. falciparum challenge after immunization with the alum-adjuvanted recombinant protein R32tet32 in a prior study. Three separate experimental challenges were conducted with 10 volunteers 2 to 4 weeks after the third dose of vaccine. The four best responders, on the basis of antibody levels (6 to 26 ,ug/ml), were challenged with two infectedmosquito bites, but only one of four immunized volunteers and one of three malaria-naive controls became parasitemic. In a second challenge study using five infected-mosquito bites as the challenge dose, three of three malaria-naive control volunteers and two of three immunized volunteers developed malaria. The third vaccinee was apparently completely protected. In the third and last challenge, three of three controls and five of five vaccinees became infected. Sera obtained on the days of challenge inhibited sporozoite invasion of hepatocytes variably in vitro (range, 45 to 90%o inhibition), but the degree of inhibition did not correlate with protection. Although antibody against the CS repeat region may protect some individuals against experimental challenge, this protection cannot be predicted from antibody levels by current in vitro assays. The functionality and fine specificity of anti-CS antibody are probably critical determinants.

diphtheria toxin, diphtheria cross-reacting material protein, meningococcal outer membrane proteins, and Pseudomonas aeruginosa toxin A as carrier molecules for either the synthetic repeat peptides (NANP)3 and (NANP)6 or the recombinant protein R32LR, which comprises the sequence [(NANP)15(NVDP)]2LR (1, 10). Preclinical evaluation of these preparations demonstrated that all were highly immunogenic in rabbits. Preliminary phase 1 safety and immunogenicity studies conducted with malaria-naive volunteers demonstrated that the most immunogenic preparation in humans appeared to be R32LR conjugated to detoxified Pseudomonas toxin A (R32Tox A). Subsequent dose-response studies, again conducted with malaria-naive human volunteers, resulted in the selection of a 400-,ug dose of R32Tox A (which induced seroconversion of 21 of 22 recipients) for further study (12a). This report details further safety, immunogenicity, and efficacy evaluation of alumadjuvanted R32Tox A.

Attempts to develop a Plasmodium falciparum malaria vaccine directed at the sporozoite stage have thus far been based on the observation that the majority of sporozoiteneutralizing antibodies produced in animals and humans appear to be directed against the B-cell epitopes represented within the repeat region of the circumsporozoite (CS) protein (3, 14). Safety, immunogenicity, and efficacy testing of the alum-adjuvanted recombinant protein R32tet32 {[(NANP)l, (NVDP)]2 fused to an Escherichia coli peptide} and the alum-adjuvanted synthetic peptide (NANP)3 conjugated to tetanus toxoid demonstrated that these preparations were safe, though poorly immunogenic (2, 8). Nonetheless, in each study, the one individual with the highest antibody response was completely protected against experimental P. falciparum challenge. Efforts to improve the humoral immune response to the repeat epitopes proceeded with the evaluation of new adjuvant preparations (11) or combinations of potent immunomodulators and delivery systems (6). Efforts to optimize carrier selection, peptide-to-carrier ratios, and peptide orientation, as well as concern over possible carrier-mediated suppression associated with tetanus toxoid-conjugated preparations in tetanus-immune volunteers, led to the systematic evaluation of cholera toxin,

*

MATERIALS AND METHODS

Study subjects. Healthy men and women between the ages of 18 and 50 years were recruited from the local community under a protocol approved by the Johns Hopkins Joint Committee on Clinical Investigation and the Army Surgeon General's Human Subjects Research Review Board. After

Corresponding author. 1834

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SAFETY AND IMMUNOGENICITY OF A P. FALCIPARUM VACCINE

providing written, informed consent, volunteers underwent baseline evaluations which included a medical history (with special attention to any previous history of malaria or splenectomy), physical examination, and routine standard laboratory tests consisting of a complete blood count, serum chemistry tests, urinalysis, and serological evaluation to exclude acute or chronic hepatitis B virus or human immunodeficiency virus type 1 infection. Volunteers were excluded from participation if they had been in a malarious area within 1 year before the start of the study; previously had malaria; had a splenectomy; had any evidence of cardiovascular, hepatic, renal, or immunologic dysfunction; or were taking any immunosuppressive medications. Hemoglobinopathy screens were performed to rule out the sickle cell trait. Twenty-one volunteers were initially enrolled in the study. After completion of the immunization series, vaccinees were recruited to participate in the evaluation of vaccine efficacy on the basis of a laboratory-based challenge study; 10 vaccinees consented. Nine unimmunized malarianaive volunteers were recruited to serve as infectivity controls during the challenge portion of the study. Vaccine preparation. The vaccine was produced by a collaborative effort between the Walter Reed Army Institute of Research, the Swiss Serum and Vaccine Institute, and SmithKline Beecham. R32Tox A consists of the recombinant-produced protein R32LR chemically conjugated to purified, detoxified Pseudomonas toxin A. R32LR was produced by SmithKline Pharmaceuticals and consists of the amino acid sequence [(NANP)15(NVDP)]2LR. Pseudomonas toxin A was purified essentially as previously described (5) and then detoxified by addition of adipic acid dihydrazide (Fluka AG, Buchs, Switzerland) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 22°C and a pH of 4.8. The resulting toxin A-adipic acid dihydrazide intermediate was then dialyzed against 0.05 M phosphate-buffered saline, pH 7.4, and any particulates were removed by centrifugation at 5,000 x g for 10 min. Additional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was then added to the toxin A-adipic acid dihydrazide intermediate; R32LR was then added, and the mixture was incubated at 22°C and pH 4.8 for 3 h. The resultant solution was filter sterilized under aseptic conditions and concentrated by evaporation under reduced pressure. The R32Tox A conjugate was then purified by sterile Sephadex G-75 column chromatography, filter sterilized through a sterile 0.22-,um-pore-size Millipore filter, and stored at 4°C. The product had an average R32LR-to-toxin A molar ratio of 6.6:1, and the acute toxicity of toxin A was attenuated over 500-fold in a murine test system. The final vaccine preparation was delivered at a concentration of 800 ,ug/ml adsorbed to aluminum hydroxide (0.4% wt/vol) with Merthiolate (0.01%) as a preservative. Immunization schedule. Volunteers received 400 ,ug of alum-adjuvanted R32Tox A as a 0.5-ml deltoid intramuscular injection at 0 and 8 weeks. Volunteers were observed for 15 min after each dose for immediate reactions and evaluated at 24 and 48 h for symptoms of headache, fever, chills, malaise, local pain, erythema, warmth, induration, lymphadenopathy, or other complaints. Nineteen volunteers received the second dose of vaccine at 8 weeks. On the basis of the safety and immunogenicity data collected after the first two doses of R32Tox A, the decision was made to proceed to an efficacy trial with a challenge model using laboratory-reared and infected mosquitoes. All available vaccinees received a third dose of vaccine administered 11 to 13 months after the initial dose. Ten vaccinees consented to malaria challenge,

1835

which was carried out 2 to 4 weeks after the third vaccine dose. Mosquito species and parasites. Anopheles stephensi mosquitoes were infected by feeding on cultured P. falciparum NF54 gametocytes via Baudruche membranes (4). Mosquito batches were sampled before use in the challenge and were used if at least 50% were infected and had at least 2+ sporozoite gland burdens. Challenge procedure. Two to four weeks after the third dose of R32Tox A, each of the challenge volunteers, along with nonimmunized controls, was exposed by placing screened containers containing the infected mosquitoes in contact with exposed skin for 5 min. Mosquitoes imbibing a blood meal were dissected to document the presence of sporozoites and to assign a sporozoite gland score. Subsequent containers of mosquitoes were then administered until all volunteers received the required number of infectedmosquito bites. Because of the concern that previous experimental challenges conducted with the bites of five infected mosquitoes were unrealistically severe and may have overwhelmed any vaccine-induced immunity, the first four challenge volunteers and three nonimmunized control volunteers were challenged with two infected-mosquito bites each. The failure of two of three control volunteers to develop patent malaria infections resulted in the two remaining groups of volunteers undergoing challenge with five infected-mosquito bites. All challenged individuals had Giemsa-stained thick blood smear examinations daily beginning on day 5 postchallenge. Antimalarial therapy with oral chloroquine was initiated when parasites were first seen on a thick smear, typically when parasites reached a concentration of 10 to 30 parasites per p.1 of whole blood. All infected volunteers were cured with a single course of therapy totalling 1,500 mg of chloroquine base. Serological assays. Immediately before and 14 days after each immunization, serum was obtained for evaluation of antibody responses. In addition, sera were obtained from challenge volunteers on the day of challenge. Sera were frozen at -70°C until analysis. Anti-CS repeat antibodies were measured by enzymelinked immunosorbent assay (ELISA) with R32LR as the solid-phase antigen as previously described (2). The concentration of specific immunoglobulin G (IgG) antirepeat antibody present in the sera was calculated by comparison to a known standard run concurrently with the test sera and is reported as micrograms of anti-R32 IgG per milliliter of serum. Anti-R32 antibody avidity was measured by a modification of the sodium thiocyanate (NaSCN) elution technique as described elsewhere (11). Immunofluorescence assays (IFAs) for sporozoites were performed as previously described (2). Fluorescence was graded from 0 to 4+, where 0 indicates no detectable fluorescence and 4+ indicates intense fluorescence over the entire surface of the sporozoite. Results are reported as the highest dilution which produced 2+ fluorescence. The ability of antisera to mediate the CS precipitation (CSP) reaction was measured with freshly dissected sporozoites as described by Vanderberg et al. (13). IgG from volunteers undergoing challenge was purified by the Affi-Prep protein A chromatography matrix system (BioRad Laboratories, Richmond, Calif.) and assayed for inhibition of sporozoite invasion (ISI) of HepG2A-16 human hepatoma cells in vitro at 2-, 20-, and 100-,ug/ml concentrations by previously described methods (9). ISI assays were

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FRIES ET AL.

INFECT. IMMUN. TABLE 1. Antibody responses observed after two doses of R32Tox A IFA resultCSP reactivity' at wk 10 0 2+ 4+

ELISA result (p.g/ml)a at wk:

4534 1439 4618 2413 9109 6617 9413 1467 4150 1687 4447 9142 4560 9271 48 9172 1527 4727 6922 2412

Geometric mean + SD

0

2

8

10

16

0 0 2 1 0 1 0 1 0 0 0 0 1 0 0 1 0 0 1 0

2 135 129 3 107 14 39 15 28 15 2 2 1 7 3 11 4 1 2 1

6 44 39 3 54 6 30 22 9 8 2 1 1 2 3 5 0 3 1 0

61 43 41 40 33 29 26 21 18 14

NDd

14 9 7 7 6 4 4 4 2 2

30 16 8 23 10 21 17 14 6 15 5 2 5 2 5 2 3 2 1

0.2 ± 3.3

7.5 ± 5.1

5.0 ± 4.0

10.6 ± 3.0

6.5 ± 2.8

200 200 200 100 200 100 100 100 200 100 0 100 200 50 50 0 50 0 0 0

23 25 25 25 25 24 24 25 25 25 17 23 25 ND ND ND ND ND ND ND

2

1 1 2 2

6

a ELISA results are reported as micrograms of IgG specific for R32LR per ml. IFA results are reported as the highest dilution giving a 2+ reaction. c The degree of CSP is reported as the number of sporozoites with the indicated scores observed, where 0 = no CSP reactivity detectable, 2+ = a granular precipitate on the surface of the sporozoite, and 4+ = a long threadlike filament at one end of the sporozoite. d ND, not determined. b

performed with IgG purified from sera obtained on the days of challenge from each volunteer. RESULTS

Twenty-one individuals, consisting of 8 males and 13 females, (mean age, 33 years; range, 20 to 47 years) participated in this study. The vaccine was generally well tolerated. The major side effect reported was local tenderness at the site of injection, which generally resolved within 48 h. Fifty percent of the volunteers developed mild erythema, induration, and warmth at the injection site after the first dose of vaccine; 39% developed these complaints and/or findings after the second dose, and the overall severity of symptoms or signs did not increase. One volunteer developed induration at the injection site and complained of pain radiating down the arm after the first injection; these symptoms resolved over 72 h. Within 12 h of receiving the second dose of vaccine, this volunteer again experienced pain radiating down the arm associated with significant erythema, induration, warmth, and tenderness at the injection site; no fever or laboratory abnormalities were noted. The volunteer was treated with analgesics and local application of heat; the complaints were resolved after 96 h. This volunteer was excluded from further immunization. A second volunteer withdrew from the study for reasons unrelated to the vaccine after receiving one dose of vaccine, and one female vaccinee was excluded from further participation in the study after becoming pregnant during the 9th or 10th week of the study. No volunteer developed biochemical or hematological abnormalities during the immunization portion of the study. Table 1 and Fig. 1 summarize the antibody response to R32Tox A as measured by ELISA, IFA, and CSP reactivity. IFA and CSP assays were performed with sera obtained

during week 10 (2 weeks after the second dose). Two weeks after the second dose, 11 of 20 volunteers evaluated (55%) had attained antibody levels greater than or equal to the antibody concentration measured (by the same assay) in the prior volunteer immunized with alum-adjuvanted R32tet32 and subsequently protected from experimental challenge (9.8 ,ug/ml). An additional four volunteers (20%) developed

1000

R32-specific IgG, ug/ml

100

A

10

A

&4, A A

AA

A

A

0.1

A 0

A

A

A

A

5

10

20 15 Week of Study

PrePostThird dose

FIG. 1. R32-specific IgG responses to R32Tox A vaccine. A

400-,ug dose of vaccine was administered at week 0, at week 8, and again after 44 to 60 weeks. Individual sera are indicated by open triangles, and the geometric mean values at each time point are indicated by the small squares and solid line. Levels of zero were arbitrarily assigned a value of 0.1 for purposes of calculation and display on a logarithmic scale.


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TABLE 2. Summary of R32Tox A challenge results Volunteer no.

Cohort 1 6617 9172 9142 4447 11879 13452 13453

Cohort 2 2412 48 9271 9136 9866 13792 Cohort 3 4447c 6617C 1687 4618 1527 14075 3354 1358

No. of infectious bites

Day of patency

% ISI with purified IgG (20 p.g/ml)

Antibody avidity index"

23 23 26 6 0 0 0

2 2 2 2 2 2 2

>30 16 >30 >30 13 >30 >30

20 59

1.029 1.214 1.127 1.964

Imm Imm Imm Ctl Ctl Ctl

2 2 4 0 0 0

5 5 5 5 5 5

11 15 >30 11 13 14

29 44 0 0 0 0

Imm Imm Imm Imm Imm

7 15

5 5 5 5 5 5 5 5

9 11 9 11 9 10 9 9

0 6 16 5 33 0 0 0

5

>30

Statusa Imm Imm Imm Imm

Ctl Ctl Ctl

Ctl Ctl Ctl

R32-specific IgG (p.g/ml)

9 9 4 1 4 1

9.8 Imm 14d a Imm, immunized; Ctl, control (unimmunized).

1.144 1.221 1.38

2.124 1.474 1.441

0.959

"Molar concentration of NaSCN necessary to reduce antibody binding in ELISA by 50%. c Volunteers 6617 and 4447 were rechallenged during experiment 3. These two volunteers did not receive an additional booster injection after their first challenge. As a result, antibody titers listed for the day of challenge for cohort 3 reflect the natural decay in antibody levels in these individuals during the 4-month period between their first and second challenges. d Volunteer 14 (not a member of any cohort) had previously been vaccinated with R32tet32 and was protected against experimental challenge. Results are from serum obtained on his original day of challenge and run concurrently with the other sera shown above for comparative purposes.

antibody concentrations in the 6- to 9-,ug/ml range by 2 weeks after the second dose of vaccine. IFA titers generally paralleled ELISA results. Only one volunteer developed serum antibodies capable of inducing a CSP reaction after two doses of vaccine. Interestingly, this volunteer required two doses of vaccine before seroconverting and did not recognize sporozoites by IFA at a dilution of 1:50. On the basis of the findings described above, it was decided to proceed with a third dose of vaccine approximately 1 year after the initial injection, followed by a mosquito-borne P. falciparum challenge for consenting subjects. The third dose was well tolerated by all recipients. As shown in Fig. 1, the third dose resulted in at least a doubling of the specific IgG level in 10 of 18 (56%) recipients, with an increase in the geometric mean repeat region-specific IgG level from 4.4 to 10.5 ,ug/ml. A total of 10 vaccinees subsequently consented to challenge, the results of which are shown in Table 2. Volunteers 6617, 9172, 9142, and 4447 received their third dose of vaccine during week 44 of the study. Two weeks later, they and three malaria-naive control volunteers underwent challenge with a total of two infected-mosquito bites each. At the time of challenge, the vaccinees had 23, 23, 26, and 6 jig of R32-specific IgG per ml, respectively, as measured by ELISA. Only one of three malaria-naive controls developed patent malaria infection, which precluded evaluation of vaccine efficacy in this group. Volunteers 2412, 48, and 9271 received their third dose of

vaccine during week 50 of the study. Volunteer 2412 did not develop a substantial antibody response even after three doses of vaccine; volunteers 48 and 9271 failed to demonstrate any boost in antibody levels in response to their third dose of vaccine, compared with their previous peak levels of 7 and 6 ,ug/ml, respectively, measured 2 weeks after the second vaccine dose. Challenge of these three vaccinees and three malaria-naive controls was conducted with the bites of five infected mosquitoes each. The three control volunteers developed patent infections on days 11, 13, and 14. Volunteer 2412, a vaccine nonresponder, developed malaria on day 11, and volunteer 48 developed malaria on day 15. Volunteer 9271 failed to develop malaria. Vaccinees 1687 and 1527 received their third dose of vaccine during week 58 and vaccinee 4618 received the third dose during week 60 of the study. These three volunteers, along with vaccinees 4447 and 6617 from the first challenge cohort and three additional malaria-naive controls, underwent challenge with five infected-mosquito bites each. All eight individuals developed patent infections between days 9 and 11. Results of ISI assays using purified IgG from selected day-of-challenge sera and avidity index determinations are summarized in Table 2. None of the IgG samples demonstrated inhibitory activity at an IgG concentration of 2 ,ug/ml. In comparison, purified IgG from the previously protected volunteer immunized with R32tet32 had an IgG concentration that resulted in a 50% ISI of 3.0 p.g/ml (7). Variable

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FRIES ET AL.

degrees of inhibition were noted at an IgG concentration of 20 p,g/ml but did not predict protection or correlate with the day of patency. Nonspecific inhibition of sporozoite invasion at an IgG concentration of 100 ,ug/ml precluded analysis. In all volunteers tested, antibody avidity exceeded that of the previously protected individual used as a control.

DISCUSSION Early experience with recombinant and/or synthetic peptide conjugate vaccines designed to induce antibodies directed against the repeat region of Plasmodium sp. CS proteins has demonstrated that these regions are relatively poor immunogens when delivered adjuvanted with alum or as tetanus toxoid conjugates (2, 7, 8). Efforts to improve the humoral immune response in humans have included expression of R32, i.e., [(NANP)15(NVDP)]2, with heterologous T-helper cell epitopes present in an 81-amino-acid derivative of the nonstructural protein 1 of influenza A virus (R32NS1), administration of the recombinant protein R32NS1 delivered with a novel adjuvant containing the cell wall cytoskeleton of mycobacteria and monophosphoryl lipid A emulsified in squalene (11), and administration of R32NS1 encapsulated with monophosphoryl lipid A in liposomes (6). These formulations have demonstrated the ability to improve antibody production in humans; however, data regarding their efficacy are still unavailable. In this study, we have shown that use of detoxified Pseudomonas toxin A as a conjugate carrier for the recombinant molecule R32LR is safe and greatly improves the humoral immune response to the P. falciparum CS repeat region. Antibody concentrations, as measured by ELISA, exceeded or approached that of a previously immunized and protected individual in 15 out of 20 (75%) of evaluable volunteers 2 weeks after they received their second dose of vaccine. At the time of challenge, all volunteers examined possessed antibodies with a higher avidity for the recombinant molecule R32LR than those of the previously protected volunteer, and six had equivalent or greater absolute concentrations. Despite the excellent results of toxin A conjugation, we were unable to improve upon the previously reported poor protective efficacy of other CS protein repeat region vaccines in this challenge model. Interestingly, the level of R32-specific IgG measured by ELISA, antibody avidity, and ISI activity demonstrated by our single protected vaccinee were all exceeded in a number of vaccinees who were not protected. While any conclusions based on this single observation must be tentative, these findings suggest that current in vitro measures of CS protein repeat region antibody are inadequate to predict efficacy. It seems likely that subtle differences in antibody function or fine epitope specificity not reflected in the available assays may be important. Alternatively, unidentified nonantibody effector mechanisms may be evoked by CS repeat region vaccines in a minority of vaccinees. Our experiences do suggest, in congruence with prior challenge trials, that some degree of protection is possible with CS protein-based vaccines and that these antigens should be included in any future multicomponent malaria vaccine. Our ability to assess protective efficacy was also impaired by the intrinsic variability of the challenge model, as evidenced by the wide range of prepatent periods observed and the steep infectious dose-response curve between two and five apparently infectious mosquito bites, a finding consonant with results reported by others (12). The relevance of

INFEC-r. IMMUN.

the current challenge model to P. falciparum exposures experienced in the field setting remains speculative. Although the model continues to represent a potentially useful tool, the aforementioned problems suggest that definitive assessment of the efficacy of this and other P. falciparum vaccines will eventually require immunization of large volunteer cohorts followed by careful prospective study under conditions of natural exposure. To this end, limited field trials of R32Tox A have been performed in Thailand and Kenya and are currently being analyzed. The results of these studies may further help to evaluate the utility of the human malaria challenge model. ACKNOWLEDGMENTS This work was supported in part by U.S. Army Medical Research and Development Command contract DAMD 17-88-C-8082. J. C. Beier is supported by NIH grant R22-AI-29000. We thank Martin Blair for assistance in preparing the manuscript and Judy Shahan for contributions to the clinical trial. REFERENCES 1. Ballou, W. R. 1991. Progress in malaria vaccines, p. 373-380. In S. J. Cryz (ed.), Vaccines and immunotherapy. Pergamon Press, New York. 2. Ballou, W. R., S. L. Hoffman, J. A. Sherwood, M. Hollingdale, F. Neva, W. T. Hockmeyer, D. M. Gordon, R. A. Wirtz, J. F. Young, R. Reeve, C. L. Diggs, and J. D. Chulay. 1987. Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet i:1277-1281. 3. Ballou, W. R., J. Rothbard, R. A. Wirtz, D. M. Gordon, J. S. Williams, R. W. Gore, I. Schneider, M. R. Hollingdale, R. L. Beaudoin, W. L. Maloy, L. H. Miller, and W. T. Hockmeyer. 1985. Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum. Science 228:996-999. 4. Burkot, T. R., J. L. Williams, and I. Schneider. 1984. Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate. Trans. R. Soc. Trop. Med. Hyg. 78:339341. 5. Cryz, S. J., Jr., E. Furer, and R. Germanier. 1983. Protection against Pseudomonas aeruginosa infection in a murine burn wound sepsis model by passive transfer of antitoxin A, antielastase, and antilipopolysaccharide. Infect. Immun. 39:10721079. 6. Fries, L. F., D. M. Gordon, R. L. Richards, J. E. Egan, M. R. Hollingdale, M. Gross, C. Silverman, and C. R. Alving. 1992. Liposomal malaria vaccine in humans: a novel, safe, and potent adjuvant strategy. Proc. Natl. Acad. Sci. USA 89:358-362. 7. Gordon, D. M., T. M. Cosgriff, G. F. Wasserman, C. Silverman, W. R. Majarian, and J. D. Chulay. 1990. Safety and immunogenicity of a Plasmodium vivax sporozoite vaccine. Am. J. Trop. Med. Hyg. 42:527-531. 8. Herrington, D. A., D. F. Clyde, G. Losonsky, M. Cortesia, J. R. Murphy, J. Davis, S. Baquar, A. M. Felix, E. P. Heimer, D. Gillessen, E. Nardin, R. S. Nussenzweig, V. Nussenzweig, M. Hollingdale, and M. Levine. 1987. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature (London) 328:257-259. 9. Hollingdale, M. R., A. Appiah, P. Leland, V. E. do Rosario, D. Mazier, S. Pied, D. A. Herrington, J. D. Chulay, W. R. Ballou, T. Derks, S. H. Yap, R. L. Beaudoin, and J. P. Verhave. 1990. Activity of human volunteer sera to candidate Plasmodium falciparum circumsporozoite protein vaccines in the inhibition of sporozoite invasion assay of human hepatoma cells and hepatocytes. Trans. R. Soc. Trop. Med. Hyg. 84:325-329. 10. Que, J. U., S. J. Cryz, Jr., R. Ballou, E. Furer, M. Gross, J. Young, G. F. Wasserman, L. A. Loomis, and J. C. Sadoff. 1988. Effect of carrier selection on immunogenicity of protein conjugate vaccines against Plasmodium falciparum circumsporozoites. Infect. Immun. 56:2645-2649. 11. Rickman, L. S., D. M. Gordon, R. Wistar, U. Krzych, M. Gross, M. R. Hollingdale, J. E. Egan, J. D. Chulay, and S. L. Hoffman.

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1991. A novel adjuvant containing cell wall skeleton of mycobacteria, monophosphoryl lipid A, and squalene substantially improves the immunogenicity in humans of a malaria circumsporozoite protein vaccine. Lancet i:988-1001. 12. Rickman, L. S., T. R. Jones, G. W. Long, S. Paparello, I. Schneider, C. F. Paul, R. L. Beaudoin, and S. L. Hoffman. 1990. Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans. Am. J. Trop. Med. Hyg. 43:441-445.

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12a.Sadoff, J. C., et al. Unpublished data. 13. Vanderberg, J. R., R. S. Nussenzweig, and H. Most. 1969. Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei. V. In vitro effects of immune serum on sporozoites. Mil. Med. 134:1183-1190. 14. Zavala, F., A. H. Cochrane, E. H. Nardin, R. S. Nussenzweig, and V. Nussenzweig. 1983. Circumsporozoite proteins of the malaria parasites contain a single immunodominant region with two or more identical epitopes. J. Exp. Med. 157:1947-1953.